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Estrogen receptor interaction with a transcription factorRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, In Vivo Diagnosis Or In Vivo Testing, Testing Efficacy Or Toxicity Of A Compound Or Composition (e.g., Drug, Vaccine, Etc.)Estrogen receptor interaction with a transcription factor description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060051295, Estrogen receptor interaction with a transcription factor. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The invention relates to a method to measure a direct interaction between an estrogen receptor (ER) and a transactivation protein in an in vitro system by providing for a detection parameter proportionally related to the degree of the interaction. [0002] Several important medicinal compounds have their therapeutic effects in an organism by an influence on transcription factors. In particular, this mechanism of action is used by those medicines, which act by mimicking steroidal hormonal actions, such as certain contraceptives and anti-inflammatory compounds. Transcription factors are proteins controlling the read-out of genetic DNA material in the cells of an organism. It is this mechanism of control which determines many properties of a living cell. The control of expression of genes in the cells can be permanent or can be transient. Transient changes are changes in response to changing environments of the cell. The presence of other cell surfaces and other molecules in the environment of the cell are a source of signals to a cell and lead to a change of its functioning. The transcription factors play an essential role as mediators of external influences on the cell. In this sense, transcription factors are part of signaling mechanisms. [0003] Well known transcription factors are nuclear receptors, such as receptors for the steroid hormones estradiol, progesterone, cortisol etc. Another transcription factor family is referred to as the nuclear factor .kappa.B (NF-.kappa.B) transcription factor family. Transcription factors of the NF-.kappa.B family are dimers composed of two subunits out of the group RelA(=p65), RelB, c-Rel, P50 and P52. A signaling system, based on mobilisation of NF-.kappa.B, operates by responding to cytokine molecules from outside the cell via an inhibiting protein I.kappa.B. The inhibitor of NF-.kappa.B, I.kappa.B, forms inactivating complexes with NF-.kappa.B, but these complexes are susceptible to enzymatic removal of I.kappa.B by phosphorylations in response to cellular exposure to cytokines. The enzymatic removal of this inhibitory factor from I-.kappa.B/NF-.kappa.B complexes make NF-.kappa.B available for activity on the genome. Another distinct transcription factor family, c/EBP-.beta., will be mentioned later in this description. [0004] Transcription factors operate as complexes comprising multiple subunits. These complexes have affinity for a domain in the genome, which domain is called a response element. One or more response elements in a DNA chain form a location from which the bound transcription factor interacts with a larger portion of the genome, the promotor region. The complexation of the transcription factor with response elements and a promotor region in DNA determine the rate of transcription of the gene linked downstream to the promotor on the DNA. It is striking that many transcription factors are binding to DNA as dimers. Thus, the nuclear steroid hormone receptors are usually composed of two identical or closely related subunits. In modern biochemical theories, the notion that transcription factors belong to separate classes without mutual interaction is now obsolete and replaced by the notion that transcription factors interact across classes of transcription factors. Thus, selective binding interactions were demonstrated between the glucocorticoid steroid receptor monomer (GR) with RelA (Harnish, Endocrinol. 141: 3403-3411, 2000). High affinity binding was discovered between androgen receptor (AR) with RelA (Panet-Raymond et al., Mol Cell Endocrin., 167, pp 139-150, 2000). The estrogen receptor a monomer is shown to form complexes with c-Jun (Teyssier et al, J Biol Chem 276 pp 36361-36369, 2001). It is believed that such complexes are building blocks of complexes which function as transcription factors or, alternatively, absorb an amount of an available transcription factor, thereby encapsulating the factor into an inactive complex (Ray and Prefontaine Proc. Natl. Acad. Sci. 91: 752-756, 1994; Kalkhoven et al. J. Biol. Chem. 271: 6217-6224, 1996) [0005] Since the signaling mechanisms have mutual influences owing to above-mentioned direct protein-protein interactions between the transcription factor subunits, the terms cross-talk and transrepression are used to describe those mutual influences. Transrepression refers to the negative influence of one signaling pathway on another. The present invention more specifically focusses on the interactions between a member of the estrogen receptor family with an NF-.kappa.B protein and the use of the interaction for manufacture of medicines. [0006] Several studies exist that show efficient repression of NF-.kappa.B signaling by ER (Quaedackers et al. Endocrinology 142:1156-1166, 2001). The mechanism underlying the transactivating and transrepressing effects of ER on NF-.kappa.B signaling is not clear. There is a possibility that the nuclear receptor and NF-.kappa.B may associate into large multimeric complexes. Such complexes can contain nuclear receptor, NF-.kappa.B and several other nuclear (co-)factors and may still be capable to bind the DNA. An example of a concept of reciprocal interaction between a nuclear receptor and an NF-.kappa.B signaling pathway is depicted in FIG. 1 [0007] (FIG. 1) Schematic representation of reciprocal transrepression by ER and NF-.kappa.B proteins. For other nuclear receptors, such as GR and PPAR.gamma., similar kinds of functional interactions with NF-.kappa.B have been described. It is at present not yet clear whether formed complexes of NF-.kappa.B with nuclear receptors consists of monodimers or dimers. [0008] The present invention provides for a method to measure a direct interaction between an estrogen receptor (ER) and a transactivation protein in an in vitro system by providing for a detection parameter proportionally related to the degree of the interaction, characterised in that the transactivation protein is c-Rel. [0009] Thus far, only weak protein-protein interaction between ER and certain types of NF-.kappa.B was reported (Stein and Yang, Mol. Cell. Biol. 15: 4971-9, 1995; Speir. Circ. Res. 87: 1006-1011, 2000) and this interaction was independent from the presence of an ER ligand. In contrast, the interaction between estrogen receptor and c-Rel described here is dependent on the presence of a compound that binds to the estrogen receptor and by doing so can induce the interaction between the estrogen receptor and c-Rel. [0010] It is useful to measure the interaction between an estrogen receptor (ER) and c-Rel, and also the influence of a compound thereon since ER/NF-.kappa.B interactions are involved in certain effects of estrogens on the cardiovascular system, bone and the central nervous system (CNS). These are three major tissues where estrogens display beneficial effects in medical treatments. An additional role of ER/NF-.kappa.B interactions in progression of breast cancer has been suggested (Rodriguez et al. Immunogenetics 39: 161-167, 1994; Nakshatri et al. Mol. Cell. Biol. 17: 3629-3639, 1997), indicating a role of NF-.kappa.B inducible genes in malignancy and chemotherapeutic resistance. [0011] Although knock-out phenotypes do not show clear functions associated to one of the NF-.kappa.B types only (Attar et al., Cancer Biol 8: 93-101, 1997), the absence of both p50 and p52 in double knock-out mice results in osteopetrosis, supporting the role of NF-K in bone metabolism (Iotsova et al., Nat. Med. 3: 1285-1289, 1997). [0012] In bone, NF-.kappa.B proteins play an important role in modulation of inflammatory genes (e.g. cytokines) and cell adhesion molecules. Both types of modulatory molecules are important teamplayers in bone homeostasis. In particular cytokines, such as Il-1.beta., IL-6 and TNF.alpha., stimulate osteoclast precursors and mature osteoclasts and thus enhance bone resorption (Manolagas, Bone 17: 63S-67S, 1995). Estrogens contribute significantly to the maintainance of bone mass by inhibiting the production of such cytokines (Ray and Prefontaine Proc. Natl. Acad. Sci. 91: 752-756, 1994; Stein and Yang, Mol. Cell. Biol. 15: 4971-9, 1995]. The importance of NF-.kappa.B in bone metabolism is further exemplified by knock-out studies as mentioned above (Iotsova et al., Nat. Med. 3: 1285-1289, 1997). In the CNS, several reports indicate an involvement of NF-.kappa.B in various processes but its exact contribution is still unclear. Factors that are linked to synaptic plasticity, long-term memory and Alzheimer disease, have been identified as brain-specific activators of NF-.LAMBDA.B (O'Neill and Karltschmidt, Trends Neurosci. 20: 252-258, 1997; Grilli et al, J. Biol. Chem. 271: 15002-15007, 1996). In addition, inhibition of NF-.kappa.B is associated with survival of neuronal cells (Maggirwar et al. J. Neurochem. 74: 527-539, 2000, Irving et al., Neurosci. Lett. 288: 45-48, 2000). Additional associations have been made between NR/NF-.kappa.B cross-talk and the serotonin system (Wissink et al, Mol Endocrinol. 15(4):543-52, 2001), the latter being a valid potential entry for treatment of depression. In CV, estrogens are thought to inhibit the development of atherosclerosis. One of the earliest events in the development of atherosclerosis is the infiltration of monocytic cells into the vessel wall. A critical step is the recruitment of monocytes from the circulation. This recruitment involves several steps. The initial interaction between monocyte and endothelium appears to be transient, resulting in the rolling of monocytes along the vessel wall. The rolling monocytes then become activated by locally secreted factors generated by the endothelium, resulting in their arrest and firm adhesion to the vessel wall. Finally, the monocytes transmigrate the endothelium. The initial rolling interactions are mediated by the selectins (e.g. E-selectin), whereas firm adhesion and diapedesis appear to be mediated by the interaction of integrins on the surface of monocytes with immunoglobulin gene superfamily members expressed by endothelial cells (e.g. ICAM, VCAM). Expression of E-selectin, VCAM and ICAM is enhanced as a result of transcriptional activation of all three genes by cytokines via NF-.kappa.B signaling. Therefore, by interfering with this NF-.kappa.B induced expression of cell adhesion molecules, the infiltration of monocytes resulting in atherosclerosis, may be reduced. Expression of c-Rel in the blood vessel wall is clearly enhanced in response to ballooning (Landry et al., Am. J. Pathol. 151: 1085-1095, 1997), suggesting a role for c-Rel in vascular transformation. [0013] Other interesting functions of c-Rel are its function in control of programmed cell death and its role in activation of macrophages. Overexpression of c-Rel induces apoptosis in bone marrow cells, whereas it is protective against cell death in fibroblasts (Abbadie et al., Cell 75: 899-912, 1993). Macrophages from mice that are deficient for c-Rel produce higher than normal levels of inflammatory cytokines (Grigoriadis et al., EMBO J. 15: 7099-7107, 1996). [0014] The concept that c-Rel seems more crucial than RelA for ER/NF-.kappa.B transrepression is new, regarding information as present in literature. Therefore, it offers a unique opportunity for selection of compounds that selectively induce ER/c-Rel association. The assays to identify such compounds are hereby provided. A specific embodiment of the invention is a yeast two-hybrid assay. [0015] With such methods according to the invention there is also provided a method for selection, out of a number of compounds, of a compound for therapeutic efficacy in osteoporosis by carrying out the method, according to the invention as described above, repeatedly, optionally in parallel, in the presence of each of the number of compounds in the system and selecting a compound which enhances the interaction between an estrogen receptor and c-Rel. [0016] Further improvement of the selection of a compound which enhances the interaction between an estrogen receptor and c-Rel can be done in animal models, preferably an animal model for osteoporosis. [0017] Animal models include a method which monitors the capacity to prevent bone loss or stimulate bone formation in an in vivo model for osteoporosis. In this model, three month-old ovariectomised rats treated for four weeks orally with the compound to be tested, as described by Ederveen. Journal of Bone & Mineral Research. 14(11):1963-70, 1999]. Immediately after ovariectomy a four week oral treatment with various doses of the test compound is started. After four weeks of treatment trabecular bone mineral density (TBMD in mg/cm3) of the distal metaphysis of the femur is measured by pQCT (peripheral Quantitative Computed Tomography machine; XCT 960A, Stratec, Birkenfeld, Germany). A scan including a 1 mm thick slice, with a resolution of 0.148.times.0.148 mm, is taken 5 mm from the distal end of the femur. Intra- and inter-assay variation for the measurement of trabecular bone density in the distal femur is 3%. The XCT-960A is calibrated with a standard of hydroxyapatite embedded in acrylic plastic. In addition, cortical bone mineral density can be measured at the medial site of the femur. [0018] The provision of compounds which induce the direct interaction between an estrogen receptor and c-Rel in a system, provide a method to influence the interaction in a system by adding a compound to the system, which compound influences the direct interaction between an estrogen receptor (ER) and c-Rel. In a more specific embodiment of this aspect of the invention, the method to influence the interaction in a system by adding a command to the system is obtained by selecting an in vitro culture medium as the system. Most preferred is to use these methods to influence the interaction between an estrogen receptor and c-Rel by addition of a compound to the system with a compound, which influences the interaction selectively in comparison to an influence of the compound on ER transcriptional activity. [0019] Compounds which are suitable for the methods defined in the previous paragraphs can be used for the manufacture of a pharmaceutical composition for use in a therapeutic treatment to influence the interaction between estrogen receptor and c-Rel. Such a therapeutic treatment is for example, and preferably, a treatment for osteoporosis. [0020] In this description the terms have the following meaning: A direct interaction between two proteins is meant to be a physical contact based on chemical affinity under circumstances comparable to the natural environment of the proteins. However, this can not only mean an increase or a decrease in the binding between these proteins, but also a modification of the secondary structure of the binding complex, making it more or less efficacious in preventing or inducing transcriptional activity. [0021] Transactivation protein, protein in general called transcription factor that has a transactivating activity on gene expression. Transactivation proteins interact with co-factors (co-activators, co-repressors, others) and other (transcription) factors that belong to the transcription machinery and with which they together form a complex that modulates transcription initiation. [0022] estrogen receptor (ER) transcriptional activity is the transactivating activity of the estrogen receptor obtained by direct interaction of the activated ER dimer with specific ER responsive regulatory elements (ERE) gene regulatory parts of the genome. As a consequence of activation of the ER dimer it interacts with a specific set of co-activators and other cofactors, (transcription) factors and in complex with the transcription machinery activate transcription initiation. By "regulatory element" or "promoter" is meant a DNA sequence that is capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. A promoter may be linked to a heterologous reporter gene capable of signaling the activation of the regulatory element. In such a construct, the promoter influences the transcription from the heterologous gene. Suitable reporter genes are for instance luciferase, chloramphenicol acetyl transferase, beta galactosidase and secreted placental alkaline phosphatase. [0023] An estrogen receptor (ER) is a protein that binds the natural hormone 17.beta.-estradiol and belongs to the family of nuclear receptors and more specific to the subfamily of steroid receptors. The nomenclature of nuclear receptors has been described (Laudet V., Auwerx J., Gustafsson, J.-A. & Wahli, W. (1999) Cell 97 161-163). At present two closely related estrogen receptors are known, being ER.alpha. and ER.beta.. Also included are those variant and modified proteins that bind 17.beta.-estradiol and are functional equivalents of the estrogen receptor and that retain the described biological function in its interaction with c-Rel. With a "functional equivalent" is to be understood a molecule capable of excerting the same biological function as the molecule it refers to, having major structural features in common and being available as an alternative means to the skilled person on the basis of generally available technology in this field. Often, such equivalents are defined to be proteins with 90%, 95%, 98% or 99% aminoacid identity to the proteins defined by sequence data. Clearly such definitions are comprised as more specific definitions in the present definition, provided the function of the estrogen receptor equivalent in binding to c-Rel or vice versa, the c-Rel equivalent to the estrogen receptor is retained. Percentage identity is hereby defined as: Identity .function. ( % ) = Number .times. .times. of .times. .times. identical .times. .times. residues .times. .times. between .times. .times. two .times. .times. sequences Length .times. .times. of .times. .times. aligned .times. .times. sequences - Length .times. .times. of .times. .times. all .times. .times. gaps [0024] after optimal alignment of the sequences. Optimal alignment can be performed using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970)) that maximizes the number of matches and minimizes the number of gaps. 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